The present invention relates to a process for producing rhamnolipids involving culturing Pseudomonas chlororaphis strain NRRL B-30761 in a first aqueous culture medium containing about 0.3% NH4H2PO4, about 0.2% K2HPO4, about 0.2% carbon source, about 0.5 mg/L FeSO4, and about 0.1% MgSO4 for about 24–about 48 hours at about 25°–about 30° C. with orbital shaking, and then culturing Pseudomonas chlororaphis strain NRRL B-30761 in a static second aqueous culture medium containing per liter about 2% carbon source, about 0.7 g KH2PO4, about 0.9 g Na2HPO4, about 2 g NaNO3, about 0.4 g MgSO4.7H2O, and about 0.1 g CaCl2.2H2O for at least about 72 hours at about 20°–about 23° C., wherein the first and second aqueous culture medium contains only one source of carbon.
Rhamnolipids were first isolated from Pseudomonas aeruginosa in 1949 (Jarvis, F. G., and M. J. Johnson, J. Am. Chem. Soc., 71: 4124–4126 (1949)). Rhamnolipids are predominantly constructed from the union of one or two rhamnose sugar molecules and one or two β-hydroxy fatty acids (3-hydroxy) (Lang, S., and D. Wullbrandt, Appl. Microbiol. Biotechnol., 51: 22–32 (1999)). Rhamnolipids with one sugar molecule are referred to as mono-rhamnolipids while those with two sugar molecules are di-rhamnolipids. The length of the carbon chains found on the β-hydroxy portion of the rhamnolipid can vary significantly; however, in the case of Pseudomonas aeruginosa ten carbon molecule chains are the predominant form (Deziel, E., et al., Biochim. Biophys. Acta, 1485: 145–52 (2000)). Primary rhamnolipid production by P. aeruginosa occurs during stationary growth phase in rapidly agitated liquid media with limiting concentrations of nitrogen or iron at 37° C. (Guerra-Santos, L. H., et al., Appl. Microbiol. Biotechnol., 24: 443–448 (1986)). P. aeruginosa growth and rhamnolipid production can occur using a range of different primary carbon sources. The highest levels of rhamnolipid production in P. aeruginosa occurs when using vegetable based oils as carbon sources including soybean oil (Lang, S., and D. Wullbrandt, Appl. Microbiol. Biotechnol., 51: 22–32 (1999)), corn oil (Linhardt, R. J., et al., Biotech. Bioeng., 33: 365–368 (1989)), canola oil (Sim, L., et al., J. Ind. Microbiol. Biotechnol., 19: 232–8 (1997)), and olive oil (Robert, M., et al., Biotechnol. Lett., 11: 871–874 (1989)).
Rhamnolipids exhibit several promising industrial applications. Rhamnolipids are powerful natural surfactants, capable of reducing the surface tension of water from roughly 76 mN/m to between 25–30 mN/m, and are emulsifying oils (Guerra-Santos, L., et al., Appl. Environ. Microbiol., 48: 301–5 (1984)). Rhamnolipids also demonstrate significant antibacterial and antifungal activity, suggesting a role for these compounds in medical and agricultural fields (Desai, J. D., and I. M. Banat, Microbiol. Mol. Biol. Rev., 61: 47–64 (1997)). Since rhamnolipids are derived from a “natural” source and in a pure form have low toxicity levels, this serves to make the rhamnolipids an attractive alternative to more synthetic compounds. However, since rhamnolipids are produced by P. aeruginosa, a known human, animal, and plant pathogen, there are considerable safety issues that would have to be properly addressed before rhamnolipids produced in this manner would be considered safe. Since addressing these safety concerns could prove to be cost prohibitive, the effort to commercialize rhamnolipids should be helped considerably if the rhamnolipids could be produced by a non-pathogenic microbe. We have found that a bacterial strain belonging to the non-pathogenic bacterial species P. chlororaphis is capable of naturally producing rhamnolipids under conditions different from any previously described bacterial methods for the production of rhamnolipids.